Quantification of Asialoglycoprotein Receptor by Ligand Binding. Rat hepatocytes isolated by collagenase perfusion specifically and avidly bind (125I) asialoorosomucoid. The number of "functional receptors" capable of ligand binding is 500,000/hepatocyte (Kd is about 7 times 10 to the minus 9th M). The rate of ligand uptake at 37 degrees C is 0.1 pmol/min/hepatocyte. Serum or serum proteins inhibit both maximal ligand binding as well as ligand uptake by up to 90%. The nature of this inhibitory activity is unknown, but provided evidence for the underestimate of ligand binding in many studies in their presence. Isolation and Identification of Asialoglycoprotein with Monoclonal Antibody. The asialoglycoprotein receptor from rat liver has been purified via solubilization and affinity chromatography on asialoorosomucoid-sepharose. The preparation yields four distinct polypeptides of m.w. 40,000-120,000 daltons. We prepared a monoclonal antibody which both immunoprecipitates solubilized receptor activity and which blocks the binding of galactose-terminal glycoproteins to immobilized receptor. The monoclonal antibody, as well as a rabbit antireceptor antiserum, immunoprecipitated all four polypeptide species. Peptide fingerprinting of the individual (125I) labeled species demonstrated a nearly identical pattern, also suggesting that the four polypeptides have a similar primary structure. To identify and quantitate the asialoglycoprotein receptor on the hepatocyte cell surface, intact cells were iodinated with lactoperoxidase and the solubilized membranes were reacted with antireceptor antibody. The 55,000 and 65,000 m.w. species were the major species found. Our results suggest that the size of the surface receptor is at least 55,000 daltons and that it comprises between 1-2% of the iodinated hepatocyte surface protein.